DESCRIPTION: Calcific aortic valve disease (CAVD) is the most prevalent aortic valve disorder, but there is no known treatment for CAVD other than total valve replacement. Although we know much about the characteristics of severely diseased valves, relatively little is known about the early stages of CAVD, which inhibits our ability to develop pharmacological treatments for this disease. Significant limitations associated with the availability of human specimens and the physiological relevance of animal models have motivated us to propose the development of alternative approaches to studying CAVD. In this application, we describe our plan to generate engineered in vitro culture models that mimic valve features in order to identify the sequence of events in early CAVD. Specifically, we will focus on replicating three critical events in CAVD: extracellular matrix (ECM) disarray, inflammation, and lipid oxidation. Guided by the hypothesis that altered glycosaminoglycan (GAG) deposition is one of the earliest events in CAVD and is sufficient and necessary for initiation of subsequent pathological events in the CAVD cascade, we propose the following Aims: Aim 1: Evaluate the ability of the GAG enrichment to initiate elements of CAVD. In this Aim, we propose to employ innovative ECM manipulation techniques to generate 3-D valve cultures that exhibit a GAG- enriched structure that is characteristic of early CAVD. We will then investigate whether these diseased constructs are able to induce valvular interstitial cell (VIC) dysfunction, and, conversely, whether healthy constructs can rescue diseased VICs. Aim 2: Determine whether GAG enrichment is a prerequisite for progression of the CAVD cascade. We will provide 3-D VIC cultures with other known building blocks of CAVD; specifically, we will determine whether GAG enrichment is required for macrophage infiltration and lipid oxidation, two other key features of CAVD, thereby allowing us to characterize the sequence of these events. We will also determine whether an organized ECM environment has a protective effect on VICs exposed to other disease stimuli. Our novel strategies for investigating ECM regulation of cell function may be applicable to a broad range of disease investigations, and will specifically enable us to elucidate information about the sequence of events in early-stage CAVD, thereby achieving a critical first step toward developing much-needed treatments for this disease.